Screening of Leguminous Plants for VAM Association and Their Role in Restoration of Degraded Lands

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1 Screening of Leguminous Plants for VAM Association and Their Role in Restoration of Degraded Lands Kiran Bargali Department of Botany, DSB Campus, Kumaun University, Nainital, Uttarakhand , India Abstract: In present study, 50 leguminous plant species were assessed for association of Vesicular-Arbuscular Mycorrhizal fungi. For this, fine roots of these plants were carefully dug out, washed and stained using root clearing methods and observed under microscope. Out of 50 species screened, 5 showed no VAM association, 2 species showed very low level of colonization (> 20%), 17 species showed 20 to 49 % colonization, 24 species showed 50 to 69 % colonization and only 2 species showed very high level of colonization i.e. <70%. Most of the plant showed hyphae with vesicle/arbuscles. However in five species viz. Bahunia retusa, Crotolaria albida, Desmodium elegans, D. heterocarpon and Vicia rigidula only hyphae of mycorrhizal fungi is present. Thus, the legumes with high to very level of VAM colonization can be use in restoration of degraded lands. [Journal of American Science. 2011;7(1):7-11]. (ISSN: ). Keywords: Legumes, roots, vesicles, arbuscles, colonization 1. Introduction Restoration is defined as a tactic employed to return degraded lands to its original condition. Of the total earth s surface 78% of the land area is unsuitable for agriculture. Out of the remaining land, about 9% suffers from physical, chemical and biological constraints requiring special management practices. Since, land degradation process involve loss of vegetation and accelerated run off and soil loss, they require assistance for their restoration and regeneration. Mycorrhizal fungi and nitrogen-fixing bacteria are among the major beneficial components of soil microbial community, which contribute to plant growth and survival by reducing stresses through symbiosis (Sylvia and Williams 1992). These plants have special nutritional relationship between the two symbionts: the high phosphorus requirement of the nitrogen fixing root nodule and the high nitrogen requirement of the chitin walled VAM fungi and the high carbon requirement of both. Since each symbiont can supply the other s need in excess, the endophytes can bring about when the association is grown in nutrient-deficient soil (Norris et al 1994). In addition, VAM mycelia can extend to a long distance and can link the rhizosphere and mycorrhizosphere of different plant species; it can make common pool of the available nutrients of the other plant species (Norris et al 1994). This way, the nutrients released into the overlapping mycorrhizosphere by plant root exudation or by root and nodule decay become available for non-n 2 fixing plants (Simard et al 1997), which can play important role and facilitates the survival and growth of other plant species. Such observations led to a view of the legume microsymbionts as biological substitutes for fertilizers. The present study assessed VAM colonization in leguminous plants of Kumaun Himalayan region. This study has greater implications as results of present study can be used to stabilize many degraded areas in the region, which is a frequent phenomenon all over the Himalaya. 2. Material and Methods This study was conducted in Kumaun Himalayan region of Central Himalaya, India. Kumaun is situated between the latitude of 28 o o 49 N and longitudes of 78 o o 05 E covering an area of about 21,033 km 2 ranging from 300 m to the 5400 m elevation. Broadly this region can be differentiated into four physiographic domins viz. the outer Himalaya with Terai and Bhabhar belts and Siwalik ranges; the Lesser Himalaya; the Great Himalaya and the Trans Himalaya (Jalal 1988). The climate of the region is governed by the monsoon and the year can be divisible into summer (April- mid June), rainy (mid-june- September) and winter (October - Feburary). The annual rainfall is about 2500 mm in most of the places, of which about threefourth occur during rainy season (Singh and Singh 1992). 3. Material and Methods The field surveys were conducted in different forest sites and leguminous plants growing in the region were identified. For each species fine terminal roots 7

2 were collected from different places of root system selecting five plants at random and collected in separate polythene and brought to the laboratory. Roots were gently washed, cut into one cm segments and preserved in formalin-acetone alcohol (FAA) in the ratio of 90:5(v: v: v). These preserved root samples were used for studying the level of VAM colonization. Root clearing method (Phillips and Haymann 1970) was used to stain the roots by heating in KOH. The root sample stored in FAA were washed thoroughly with tape water and placed in test tubes and 10% KOH solution was added. These test tubes were heated at 90 o C for one hour in a water bath. After heating, the KOH solution was poured off from the test tube and roots were rinsed with several changes of distilled water. Then roots were placed in 1% HCL solution for 5 minute to acidify the roots. The roots were again rinsed several times. After rinsing, the roots were stained with 0.1% trypan blue staining solution and heated again for 5 minutes. Extra stain was poured off and lactophynol solution was added. Heavily pigmented roots were bleached prior to staining with alkaline H 2 O 2 (Phillips and Haymann 1970). The root segment were then pressed gently and observed under a microscope for VAM colonization. Percent VAM colonization was calculated using Nicolson simple formula (1955; see; Gupta and Mukerji 1999 ): No. of root segments colonized with VAM Colonization (%) = x 100 Total number of root segments observed The colonization was categorized in the following groups based on the intensity of infection: 1. Excellent: Mycelia/vesicle/arbuscules present on whole surface of the root bits (1 cm length) in very large number. 2. Good: Mycelia/vesicle/arbuscules present on whole surface of the root bits. 3. Moderate: Mycelia/vesicle/arbuscules present sparsely on the surface of the root bits. 4. Poor: Mycelia/vesicle/arbuscules present on root surface only in few numbers. 5. Nil: Mycelia/vesicle/arbuscules totally abscent. 3. Results A total of 50 plant species belonging to 3 families of legumes (Papilionaceae, Caesalpiniaceae and Mimosaceae) were surveyed for VAM association. Out of 50 plants sampled, 5 plants namely Crotolaria madicaginea, Lathyrus aphaca, Vicia pallida, V. tetrasperma and Pterocarpus marsupium did not possess any VAM association. Two plant species viz. Crotolaria albida and Desmodium heteropogon possessed very low level of association (less than 20%). The sparse infection in these species could be attributed to the continuous cover of root hairs. Baylis (1975) suggested that plants with greater number of root hairs would have less dependence on mycorrhiza. Intermediate level of association (20-49%) was recorded in 17 plant species. Some of the important species in this range were Astragalus leucocephalus, Lespedeza gerardiana, Bahunia variegata etc. High level of VAM colonization (50-69%) was recorded in 24 plants including Indigofera dousa, Cassia floribunda, Albezia lebbeck etc. Two plants Indigofera heterantha and Trifolium repens possessed very high (> 70%) level of colonization (Table 1). There were differences between the different species of same genus in the percentage of root infected. For example, in Indigofera dousa the infection was 64% while in I.heterantha the infection was 72% (Table 1). Five plants showed presence of only VAM hyphae in their roots where neither arbuscules nor vesicles were recorded. They were Bahunia retusa, Crotolaria albida, Desmodium elegans, D. heterocarpon and Vicia rigidula. Rest of the species contained hyphae along with vesicles and or arbuscles (Table 1). Although many species (25%) of the legume in the present study possessed arbuscules, the number of plants possessing vesicles was higher than plants bearing arbuscules. These results suggested that roots of majority of the plants colonized were mature as vesicles are storage organs and generally produced in the older region of the infection. 8

3 Table 1.Vesicular Arbuscular Mycorrhizal colonization in some leguminous plants of Kumaun Himalaya. Species Habit Distribution Habitat Percent of root segment colonized A B C Family Papilionaceae Astragalus chlorostachys Erect As an undergrowth of birchrhododendron Lindl Shrub forest A. leucocephalus Garh.ex Herb Shady and dry places Benth Argyrolobium flaccidum Prostrate Upto 2800 On the edges of miscellaneous forest (Royle) Joub. and open grassy localities A. roseum (Camb.) Joub. Prostrate Upto 2300 Common on open grassy localities Cajanus mollis (Benth.)Van Herb Upto 2000 Shady dry and sunny places der Maessen Crotolaria albida Heyne.ex Herb Upto 2000 Open grassy slopes Reth C. madicaginea Lamk. Erect Upto 1300 Open grassy places Dalbergia sissoo Roxb Tree Upto 1500 Often planted roadsides Desmodium elegans DC Shrub In oak forests and scrub jungles D. floribundum D. Don Erect Upto 2600 Common in oak-rhododendron forest and grassy localities D. heterocarpon (Linn) DC. Suberect or prostrate under shrub D. microphyllum (Thumb) DC Diffused perennial Upto 1600 Upto 2000 Common in grassy localities and forest clearing Grassy localities, roadsides and forest edges D. gangeticum Linn Shrub Upto 1500 Common in sal forest, roadsides grassy localities Dolichos fulcatus Klein Herb In grassy localities Erythrina arborescens Roxb. Tree Throughout the hills Flemingia bracteata Wight. Undershrub 1500 Common in sal forest F. strobilifera R.Br.ex Ait Shrub Upto 2500 In oak forest and grassy slopes in chir pine forest F. vestita Benth ex Baker Trailing Grassy localities throughout the hills Indigofera cassioides Rottl. Ex Shrub Common in sal, chir and DC miscellaneous forests I. dosua Buch-Hm. Ex Don Diffused In open chir and oak forests shrub or under shrub I. heterantha Wall ex Brandis Shrub Upto 3000 Throughout the hills along way sides and vacant plots in oak forest Lathyrus aphaca Linn Annual Upto 2200 Common in fields, roadsides and trailing field borders Lespedeza gerardiana Garh.ex Baker Undershrub Open grassy localities Medicago denticulata Willd. Annual procumbent M. lupulina Linn Decumbent ascending Parochetus communis Buch.- Ham ex D. Don Upto 1500 Upto 1500 Common in gardens, agricultural fields and waysides Common in grassy localities and roadsides Herb Upto 3800 Common on damp shaded plces and grassy localities Pterocarpus marsupium Roxb. Tree In open miscellaneous forests Smithia ciliata Royal Diffused In marshy and sandy localities Trifolium repens Linn Perennial In waste places, waysides and fields Trigonella. emodi Benth Herb Common on grassy fields and ground vegetation in oak forest T. gracilis Benth Diffused In forest edges and grassy slopes Vicia pallida Turoz. Climber In waste places and agricultural fields Intensity of colonization* 9

4 V. rigidula Royal Herb Forest edges, scrub jungles and roadsides V. tetrasperma (Linn) Moench Prostrate Grassy localities climbing annual V.vexillata (Linn) A Richard Trailing Upto 2500 In pine forest and open grasslands Zornia gibbosa Span. Herb Upto 1500 In grassy localities, river beds, roadsides and borders of agricultural field Family Caesalpiniaceae Bahunia retusa Buch- Tree Common in miscellaceous forests Ham ex Roxb. B variegata Linn. Tree In chir pine and miscellaceous forest Caesalpinia bonduc (Linn) Rambling Upto 1000 Common in miscellaneous forest Roxb. climber Cassia floribunda Shruby Upto 1800 Throughout the hills in waste lands Cav.Descr. C. mimosoides Linn. Prostrate Upto 1600 In grassy open localities, roadside and wayside C. occidentalis Linn Erect or Upto 1300 In waste lands, roadsides and sometimes near river banks C. tora Linn. Erect or Upto 1200 In waste lands, roadsides and agricultural field Delonix regia (Hook.) Raf. Tree Upto 1000 Planted roadside; and as ornamental plant Family Mimosaceae Acacia farnesiana (Linn.) Shrub or Upto 1800 Throughout the area Willd small tree A. nilotica (Linn) Del Small tree Upto 1400 In valleys Albizia chinensis (Osbeck) Tree Scattered in open and miscellaneous Merr. forests Albezia lebbeck (Linn.) Large tree Upto 1500 In miscellaneous forests Willd Leucaena leucocephala Tree Upto 1400 Planted (Lam.) De. Wit. Mimosa himalayana Gamble Small tree or straggling shrub Upto 1600 Along the water courses and scrub jungles M. pudica Linn. Under shrub Upto 1500 Common on roadsides and waste places A =percent external colonization; B= percent internal colonization C= percent vesicles 1= Excellent; 2= Good; 3= Moderate; 4= Poor; 5= Nil Discussions Degradation of natural resource and environment has been a global problem. In India, more than half of its geographical area faces problem of land degradation of one or the other kind. In the Himalayan region, degradation of forests has become a wide spread feature and restoration of such degraded land has become a major challenging conservational problem. Many efforts have been made to check the natural resource but have not yielded the desired sustainability. Microbial populations are key components of the soil-plant system where they are immersed in a framework of interactions affecting plant development (Lynch 1990).Perhaps the most widespread and certainly significant mutualism between plants and fungi is the root symbiosis, termed as arbuscular mycorrhiza(am). These AM fungi are the most common natural association makers with the nodulated nitrogen-fixing legumes and other plants (Kothamasi et al 2001). Woody legumes are useful for revegetation of water- deficient ecosystems that have low availability of N, P and other nutrients (Danso 1992). The scarcity of available P and the imbalance of trace elements in degraded ecosystems actually limit establishment of legumes and nitrogen fixation. But when associated with mycorrhizae it was found to increase the establishment of legumes (Barea et al 1992). In addition, woody legumes exhibit a considerable degree of dependence on mycorrhizae to thrive in stressed conditions (Osonubi et al 1991). After forming symbiotic association with legume roots, AM fungi develops an extraradical mycelium that links the 10

5 roots and soil environment and help the plants to use soil nutrients more efficiently (Barea et al 1992). Sustainable management of any degraded ecosystem involves practices that are equally concerned with productivity and soil conservation. In this situation, the Vesicular-arbuscular mycorrhizal legumes could reduce the amount of fertilizer needed for the establishment of vegetation and also increase the rate at which the desired vegetation becomes established by stimulating the development of beneficial microorganisms in the rhizosphere. Since, the cell walls of VAM hyphae are composed of amino-sugar chitin, the soil mycelium may be one of the most important vehicles for nitrogen and carbon input into the soil. Owing to their role, the VAM legumes can be used for revegetation of eroded, desertified or degraded ecosystems. In addition, the dual inoculation (Vesicular- Arbuscular Mycorrhiza along with Rhizobium) can be a biological tool for the management of N 2 - fixing plants in restoring and maintaining soil fertility. The capacity of VAM fungi to act as biofertilizers, bioregulators and bioprotectors has repeatedly been demonstrated. These associations help to maintain the general plant vigour under a variety of adverse and inhospitable ecological conditions. Present investigation is an attempt to enhance our knowledge of the ecology and applicability of VA Mycorrhizal legumes in successful reclamation and restoration practices in degraded ecosystems. On degraded sites where original soil has deteriorated markedly, these VAM associated nitrogen- fixing species can be planted. They can improve soil condition and growth of associated plant species through nutrient-rich leaf litter and biological nitrogen-fixation and therefore, useful in restoration of degraded lands. Acknowledgements: Financial support from Department of Science and Technology, New Delhi in form of SERC Fast Track Project is gratefully acknowledged. Corresponding Author: Dr. Kiran Bargali Department of Botany DSB Campus, Kumaun University Nainital, Uttarakhand , India kiranbargali@yahoo.co.in References 1. Sylvia DM, Willams SE. Vesicular-arbuscular mycorrhizae and environmental stress. In: Bethlenfalvay GJ, Linderman RG eds. Mycorrhizae and sustainable Agriculture. ASA Special Publication 54. Madison, QWI,1992: Norris JR, Read D, Verma AK. Techniques for mycorrhizal research : methods in microboilogy. Academic Press, Harcourt Brace and Company, Publishers, London, Simard SW, Perry DA, Jones MD, Myrolds DD, Durall DM, Molina R. Net transfer of carbon between ectomycorrhizal tree species in the field. Nature 1997; 388: Jalal DS. Geographical perspectives of Kumaun. In Valdiya KS, ed. Kumaun Land and People. Gyanodaya Prakash,Nainital Singh JS, Singh SP. Forest of Himalaya: Structure, Functioning and Impact of man. Gyanodaya Prakashan, Nainital, India, Philips JM, Hayman DS. Improved procedures for clearing roots and staining parasitic and vesicular arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 1970;55: Gupta R, Mukerji KG. Effect of VAM fungus on the growth and flowering of Helianthus annuus L. Var. Peredovik EC Phytomorphology 1999; Baylis G T S The magnoloid Mycorrhiza and mycotrophy in root systems derived from it; In Sanders FE, Mosse B, Tinker PB, eds. Endomycorrhizas. Academic Press, New York USA 1975: Lynch, J.M. The rhizosphere. John Wiley, New York Kothamasi D, Kuhad RC, Babu CR Arbuscular mycorrhizae in plant survival strategies; Tropical Ecology 2001;42(1) Danso SKA, Bowen GD, Sanginga N. Biological nitrogen fixation in trees in agro ecosystems. Plant and Soil 1992; 141: Barea JM, Azcon R, Azcon Aguilar C. Vesicular arbuscular mycorrhizal fungi in nitrogen fixing systems. Methods Microbiology. 1992;24: Osonubi O, Mulongoy K, Auotoye OO, Atayese MO, Okali D. Effects of ectomycorrhizal and vesicular-arbuscular mycorrhizal fungi on drought tolerance of four leguminous woody seedlings. Plant and Soil. 1991;136: Date of submission: